Conventionally, it has not yet been known that there is a gene derived from an insect and possessing the dormancy-control activity and that such a gene exhibits a biological cell-control function. We will hereunder describe the peripheral techniques of the dormancy-control activity and biological cell-control function, in order to make the understanding of the present invention easy.
Various kinds of and diversity of insects, which have been said to be on the order of 1,000,000 kinds, forcefully and vigorously inhabit the earth under the all sort of environments. These insects live in almost whole areas on the earth including the tropics, the temperature regions, the coniferous forest regions, the ice and snow regions and the deserts as well as the lakes and marshes, while they are adapted to the environments of these regions. Such a phenomenon would be nothing but the fact that the insects certainly acquire diversified functional characteristics required for forcefully living or surviving in the all sorts of environments. We would gain a great deal of information from the insect kingdom. The functional characteristics of the insect include, for instance, biophylaxis mechanisms, growth- and development-control mechanisms, wide variety of natural and synthetic substance-decomposition and—production abilities, sharp sensory functions, behavior-control mechanisms, brain-nerve mechanisms, intermediation functions or environment-adapting ability.
We can obtain valuable information, which would be useful for establishing up-to-date and creative new technologies in the future, by analyzing the environment-adaptive and energy-saving functions of these insects. Moreover, the development of a technique, which makes the most use of physiologically active substances possessing diversified functions and derived from insects, by analyzing the functions of insects would be quite significant for the human beings. Physiologically active substances, which are isolated from insects and whose structures are identified, are also quite significant as subjects of researches for developing high quality and novel products in the fields of, for instance, agriculture and medical and pharmaceutical products.
First of all, we will hereunder describe the dormancy-control function of the insect.
The insect possesses diversified functional characteristics and, in particular, the dormancy function of the insect can be said to be a noteworthy function as an admirable environment-adapting phenomenon. The meaning of the scientifically inspecting for the dormancy of the insect is as follows:                (1) The dormancy is a phenomenon in which an organism completely stops its growth during the growing process and can be interpreted as an energy-saving biological phenomenon for overcoming the unexpected harmful environments such as high temperature or low temperature seasons and/or a shortage of provisions (or food shortage).        (2) The dormancy is a phenomenon in which the organism stops its growth, in advance, through genetic control before the life of the organism is adversely affected by the living environment or through the decoding of the environmental information. Therefore, it would be recognized as a positive strategy for the environmental adaptation unlike the simple interruption of the development.        (3) If the dormancy can be controlled, such a technique may be practically quite important for the control of harmful insects and for promoting the germination of crop seeds and it would be an applied technique in the field of agriculture.        
Accordingly, if the mechanism of the dormancy control in the insect is clearly elucidated, the structure of any dormancy-control substance is determined and the functions of the substances are elucidated, the results thus obtained can be applied to the biological industries. More specifically, the results permit the free control of the growth and development of an organism valuable for the human beings and also permit the interruption of the living activities of organisms harmful to the human beings by arbitrarily inducing the dormancy of the organisms. Consequently, the dormancy control would be an important basic research from the viewpoint of the biological industries in the 21st century. In addition, the elucidation of the functions of dormancy-control substances would be quite important for the development of high quality and novel products effective in the fields of agriculture and medical and pharmaceutical products.
For instance, an Antheraea yamamai as a kind of the insects belonging to Lepidoptera falls into-dormancy in the beginning of autumn like a variety of other insects and awakes from the dormancy in April or May after staying over the winter season, in the state of dormancy. The Antheraea yamamai apparently has embryonic diapause in the egg state like the silkworm, but the larva body is almost completely formed in the egg and it falls into dormancy in this condition. Accordingly, the dormancy of this type is recognized to be a kind of the dormancy in the pre-larval state (also called “pre-larval dormancy”). There have been known not less than 40 kinds of insects falling within those of this type, represented by Lepidoptera such as gypsy moth in addition to the Antheraea yamamai and thus they should be classified as a new dormancy type one. In this respect, there has been proposed such a model that the central hormone system of the insect is not directly involved in the pre-larval dormancy of the Antheraea yamamai, but the pre-larval dormancy is controlled by the repressive factor (RF) present in the mesothorax site of the insect and that the post-larval dormancy thereof is controlled by the maturation factor (MF) present in the 2nd to 5th abdominal segments (Suzuki et al., J. Insect Physiol., 1990, 36:855-860, the disclosure of which is hereby incorporated by reference herein). The maturation factor is partially purified and is reported to be a peptide-like hormone (Naya et al., Int. Wild Silkmoth & Silk 1, 195-200, 1994, the disclosure of which is hereby incorporated by reference herein), but the repressive factor (dormancy-control substance) has not yet been isolated at all.
In addition, in respect of the silkworm, which falls into embryonic diapause, a dormancy-inducing hormone (generally, called diapause hormone) is known as an induction hormone and the hormone is a peptide hormone comprising 24 amino acid residues and having a C-terminal amide group (Imai et al. Proc. Japan. Acad., 1991. 67B: 98-101, the disclosure of which is hereby incorporated by reference herein). In the case of the insects, which fall into embryonic diapause, however, the presence of any hormone other than the foregoing one involved in the dormancy has not yet been confirmed at all. On the other hand, in the case of the insects, which have pre-larval dormancy, any dormancy-related hormone substance has not yet been discovered at all.
Other than the insect, three kinds of substances called hibernation-specific proteins are isolated from the chipmunk (Kondo et al., J. Biol. Chem., 1992, 267: 473-478, the disclosure of which is hereby incorporated by reference herein). However, these proteins have high molecular weights on the order of 27, 25 and 20 K, respectively, the blood concentrations thereof before the hibernation are lower than those observed after the hibernation and they are low during the hibernation. Moreover, there has not yet been discovered any dormancy-control substance in any mammal. For instance, in case of the embryonic diapause of the wallaby (a kind of kangaroo), the maternal pineal body thereof is considered to be involved in the dormancy, but any dormancy-control substance has not yet been isolated at all.
As has been discussed above, there has not yet been isolated, from the insect, any gene encoding a protein having a dormancy-control activity and/or any useful dormancy-control substance having a dormancy-control activity. Moreover, the hibernation-specific proteins isolated from the chipmunk suffer from such problems that they have high molecular weights and accordingly, they are liable to cause antigen-antibody reactions and that the blood concentrations of the proteins vary before and after the hibernation and they are low during the hibernation. Furthermore, there has not yet been discovered any dormancy-control substance in the mammal. Consequently, the development of a substance having a dormancy-control activity has presently been desired from the considerably wide viewpoint, including the dormancy control of the insect and the dormancy-control and growth-control of the mammal whose dormancy phenomenon has been confirmed.
Up to now, in the case of the insects, the dormancy has been classified into egg diapause, larval dormancy, pupal diapause and imaginal dormancy. However, the dormancy stage is quite complicated as will be detailed below, from the physiological-biochemical standpoint and therefore, this dormancy stage (i.e. the pre-larval dormancy stage) should endocrinologically be classified as a new stage. More specifically, there have been known insects, such as the Antheraea yamamai which does not belong to the group simply falling into the egg diapause but uniquely falls into the dormancy in the pre-larval stage and the aspen sawfly (Trichiocampus populi Okamoto) which does not belong to the group falling into the pupal diapause but belong to the group falling into the dormancy in the pre-pupal stage prior to the pupal stage. Therefore, it may be expected that the insects of these kinds include novel dormancy-control hormones and substances related thereto.
There has not yet been isolated any dormancy-control substance, which acts on not less than 40 kinds of insects represented by Lepidoptera such as Antheraea yamamai, gypsy moth, pellucid white butterfly, tent caterpillar (Malacosoma neustria testacea Motschulsky) and daimyo oak tussock moth (Liparis aurora Butler), which belong to the group falling into pre-larval dormancy (these insects of the pre-larval dormancy type are disclosed in the article of UMEYA Y., entitled “The Hibernation Phenomena in Egg State of Insects, Based on the Hibernating Egg of Silkworm”, Bulletin of the Sericultural Experiment Station (Reports from the Sericulture Experiment Station), 1946, 12: 393-481 and UMEYA Y., “Studies on embryonic hibernation and diapause in insects”, Proc. Jpn. Acad., 1950, 26: 1-9, the disclosure of which is hereby incorporated by reference herein). There has thus been strongly desired for the isolation and identification of such a dormancy-control substance, in the fields of agriculture, forestry and pharmaceutical products, and it has also been desired for the development of a manufacturing technique for economically and efficiently preparing such substances.
The original country of the Antheraea yamamai (formal Japanese name: YAMAMAYU; technical name: Antheraea yamamai Guerin-Meneville) is Japan, the rearing of the silkworm was put on record in the EDO era and has a long history. The rearing of the larvae thereof has recently become easy due to the development of artificial feeds therefor. In addition, they are in general reared in farmhouses and there are a great deal of information concerning the rearing thereof and they can easily be available. The silkworm breeds once a year and hibernates in the egg state. The larvae of the domestic silkworm Bombyx mori (Japanese name: “KAIKO”) exclusively eat mulberry leaves, while those of the Antheraea yamamai eat leaves of, for instance, Quercus acutissima, Quercus serata Thunb, Quercus dentata Thunb and Quercus variabilis Blume. The silkworm-raising farmer breeds the larvae of the domestic silkworm, while the larvae of the wild Antheraea yamamai (“YAMAMAYU”) naturally breed and grow. The hatchability of the Antheraea yamamai is very low, the rate of the silk yarn recovered from the cocoon yarn (yarn yield) is very low and the operations for recovering the cocoon yarn are quite difficult. Accordingly, the recovery thereof is very significant and has high rarity value. The value of silk yam derived from the Antheraea yamamai is estimated at 200,000 yen or even 300,000 yen per unit kg thereof and the yarn thus has rarity value to such an extent that it is referred to as “Silk diamond”. In the silk fabric whose rate of silk yarn derived from Antheraea pernyi (i.e. the wild silkworm) incorporated is high, the slippage of the yarn is inhibited and the resistance to slip-down of seams can be improved and accordingly, such fabrics have preferably been used. For this reason, the future development in the fields, which make use of the Antheraea yamamai or a large silk yarn-producing insect, will greatly be expected. Therefore, it has increasingly become important to isolate repressive factors concerning the control of the living environment for the Antheraea yamamai and to determine the structure thereof.
The reason why the dormancy-control substance of the Antheraea yamamai has not yet conventionally been able to be identified would be as follows: The presence of dormancy-control substances has been predicted in 1990 (Suzuki et al., J. Insect Physiol., 1990, 36: 855-860, the disclosure of which is hereby incorporated by reference herein), but it took a great deal of time to improve extraction methods and to select columns for the purification and accordingly, such predicted active fractions could not be isolated and purified. A principal reason for this is that the substances to be identified arc short peptides each having a very low molecular weight (hereunder also referred to as “low molecular weight peptides” and it has been unexpectedly difficult to extract them and to select an appropriate extraction column. For this reason, there have presently been desired for the establishment of a method for purifying these substances, which is required for the isolation of such dormancy-control substances from the pre-larvae and is excellent in the yield, efficiency and production cost, and can easily be operated.
Next, we will describe substances derived from insects and possessing cell growth-inhibitory functions. As physiologically active substances derived from insects, there have been known so-called living body-protective (biophylaxis) substances or antibacterial peptides, there are as many as not less than 150 kinds of such peptides and most of them have been isolated and the structures thereof have been determined. However, it is not very long since novel substances derived from insects and having anti-tumor activities were discovered and accordingly, there has been only a small amount of information concerning these novel substances.
An example of carcinostatic substances derived from insects is a peptide called Cecropin. This peptide is one isolated from Cecropia silkworm and whose structure is determined. After the determination of the structure, a variety of substances each having a structure similar to that of Cecropin have been isolated from a variety of insects and identified. It is reported that Cecropin has anti-tumor activity to cultured cells of lymphoma and leukemia (Moore, A. et al., Peptide Res., 1994, 7: 265-269, the disclosure of which is hereby incorporated by reference herein). Moreover, it is confirmed that the gene coding for Cecropin is genetically recombined into the cultured cell derived from human bladder cancer and the resulting recombined cells are injected into a nude mouse and as a result, it was found that the cells could inhibit any growth of the tumor cells (carcinostatic effect) (Winder, D. et al, Biochem. Biophys. Res. Commun., 1998, 242: 608-612, the disclosure of which is hereby incorporated by reference herein).
It is also reported that the high molecular weight protein isolated from a chrysalis of cabbage butterfly possesses strong cytotoxic activity to cancer cells such as human gastric carcinoma cells (TMK-1), can inhibit any growth of the cancer cells and ultimately exhibits such a specific physiological activity that it induces apoptosis, this protein being named Pierisin (Koyama et al., Jpn. J. Cancer Res., 1996, 87: 1259-1262; Kono et al., 1997; Watanabe et al., 1998, the disclosure of which is hereby incorporated by reference herein). This cytotoxicity finally induces apoptosis (death of cells) and accordingly, clearly indicates that the protein surely exhibits carcinostatic activity.
The protein (Cecropin) isolated from the Cecropia silkworm and the protein (Pierisin) isolated from the chrysalis of cabbage butterfly, described above, are high molecular weight physiologically active substances having molecular weights of about 4 K and 98 K, respectively. These known physiologically active substances can effectively reduce the cancer cells by inducing apoptosis in the living cancer cells, but they suffer from a problem in that it is impossible to certainly change the stage of cancer cell cycles and to thus once maintain the cancer cells in their intermitotic state (i.e. the resting cancer cells). In addition, the application of Cecropin or the like to the living body is not desirable from the practical standpoint although there has been reported that Cecropin can inhibit any growth of human cancer cells. This is because a high molecular weight protein causes an antigen-antibody reaction in the living body.
Under such circumstances, there has strongly been desired for the development of a substance capable of inhibiting any growth of cancer cells, which is a physiologically active substance derived from an insect, which has a biological cell-control function such as a cancer cell growth-inhibitory function and which never causes any antigen-antibody reaction when it is administered to a living body.